1. Field of the Invention
Here, we describe a sensitive and specific assay and kit for the detection of chemokines having activity that is upregulated by Th-1 cytokines such as IFN-γ (interferon-γ) and chemokines that upregulate the activity of Th-1 cytokines (such as IFN-γ). The invention also relates to a sensitive and novel immunoassay method and kit for detecting MIG (Monokine Induced by interferon-γ) or other chemokines whose production is upregulated by IFN-γ. Also, the invention relates to a method of assessing the effectiveness of a compound or system in inducing an immune response by detecting the induction of the expression of such a chemokine. All references mentioned in this application are incorporated herein by reference, in their entirety.
2. Description of the Background Art
The ability to assess specific immune responses is critical for understanding the immune mechanisms underlying disease, defining the types of responses to be induced by vaccination, and evaluating vaccine efficacy. Particular emphasis has been placed on the measurement of T cell mediated responses and the identification of immune markers thought to correlate with protection. In this specification and the claims that follow, the term “immune response” includes responses that modify a pre-existing immune response.
Cytokines are immune system proteins that are biological response modifiers. Monokines are chemokines secreted from monocytes. Chemokines are cytokines that have chemoattractant properties. These biological proteins are discussed, for example, in the Illustrated Dictionary of Immunology, ed. Cruse, J. M. and Lewis, R. E., CRC Press, N.Y., (1995).
In the present application and the claims that follow, an immunoreactive substance is defined as a substance to which antibodies, or immune cells, (such as T cells and NK cells) can bind, or which stimulates the production of antibodies or activate or induces T cells. That is, an immunoreactive substance can be considered as a compound capable of inducing an immune response. Although immunoreactive substances are typically thought of as whole molecules or organisms, the term “immunoreactive substance” may also be considered as that portion of a molecule or organism which elicits the immune response. For the purposes of the present specification and the claims that follow, two immunoreactive (including antigenic) substances are considered to be the same if they each share at least one common immunoreactive portion.
IFN-γ is a prototypic Th-1 cytokine produced by a variety of cells including CD4+ T cells, CD8+ T cells and NK cells (1). The importance of this cytokine in mediating protection against a number of pathogens, including parasites, bacteria, and viruses has been well established (1). IFN-γ has been known to play a central role in orchestrating a range of immunological programs which are critical for immune protection. These programs include induction of genes involved in antigen processing, upregulation of MHC Class I and Class II expression, induction of oxygen and nitrogen radicals, and stimulation of chemokine production in vitro and in vivo (1-5). Thus, in many systems, detection of IFN-γ or IFN-γ secreting cells serves a marker for the biological effects of IFN-γ activity. Accordingly, in many systems, detection of IFN-γ or IFN-γ secreting cells following exposure to antigen is frequently used to determine immunological responsiveness.
Through their ability to recruit distinct populations of leukocytes, chemokines have the ability to enhance antigen-specific immune responses. Since IFN-γ is known to regulate the production of various chemokines (6), we sought to determine if one or more chemokines could be used as a surrogate marker for antigen-specific IFN-γ production. We hypothesized that evaluating the biological effects of IFN-γ production rather than directly quantitating IFN-γ or IFN-γ producing cells per se may provide a more sensitive and reproducible means of detecting antigen-specific IFN-γ activity. Accordingly, we studied a panel of chemokines implicated in IFN-γ mediated immune responses, including Monokine Induced by interferon-γ (MIG), Interferon-γ-inducible Protein-10 (IP-10), Monocyte Chemoattractant Protein-1 (MCP-1), Macrophage Inflammatory Protein-α (MIP-α), and Regulated Upon Activation, Normal T-cell Expressed and Secreted (RANTES) (7, 8). This disclosure describes a novel assay for detecting antigen-specific MIG or antigen-specific IFN-γ or antigen-specific IFN-γ producing T-cells, based on flow cytometric quantitation of the antigen-specific, MHC-restricted, IFN-γ mediated induction of MIG expression. Our studies establish that this is a specific and sensitive assay for detecting high as well as low levels of antigen-specific IFN-γ- and/or antigen-specific IFN-γ secreting T-cells.
To date, the detection of low levels of antigen-specific cellular immune responses has been problematic, particularly in human systems. Antigen-responsive CD4+ T cells responses are routinely detected by assessment of lymphoproliferative potential or capacity to produce antigen-specific cytokines via ELISA. Antigen-specific CD8+ T cell responses are generally still evaluated by cytotoxic lysis of target cells or limiting dilution analysis (LDA), as they have been for over 15 years. However, these conventional assays are cumbersome and laborious, require extensive tissue culture and are not sensitive enough to detect low frequencies of antigen-specific cells. Furthermore, they can not be used to evaluate responses associated with cells which may respond to specific antigen by cytokine production, for example, but may not proliferate.
More recently, alternative methods have been developed to detect antigen-specific CD4+ and CD8+ T cell mediated immune responses. These assays include enumeration of cytokine producing cells at the single cell level by ELISPOT or by flow cytometry using intracellular cytokine staining techniques, or by directly quantitating peptide-specific clonotypes using tetramer technology.
In certain applications, it is desirable to evaluate the production of specific cytokines produced by antigen-specific cells. Currently, detection of antigen-specific cytokine-producing cells by cytokine-specific ELISPOT assay is gaining widespread acceptance as the most appropriate method available for detecting antigen-specific cellular immune responses. This method determines the number of cells producing a specific cytokine after in vitro culture in the presence of a specific peptide/immunogen and can be used to reproducibly detect low numbers of cytokine producing cells. The sensitivity for detecting low frequencies of responsive cells can be increased with prolonged culture and/or restimulation. However, prolonged in vitro cultivation and variations of culture conditions may not accurately reflect in vivo immunologically relevant events. Additionally, the laboratory procedure for completion of the ELISPOT assay is time consuming. Furthermore, quantitation of ELISPOT is achieved by subjective manual counting or by computerized counting microscopes which provide an objective and reproducible means of enumerating ELISPOTs, however, the cost of these machines do not make them readily available to most laboratories. Moreover, ELISPOT assays require additional time for coating plates and typically longer culture times and an additional day for development for the assay. Accordingly, there is a need in the art for a sensitive, specific, and relatively faster assay that requires short culture time prior to analysis.
Tetramer staining is more sensitive than traditional methods for detecting antigen-specific cells, but the use of this technique is restricted to well characterized epitopes in association with defined MHC alleles, and requires subsequent culture for functional characterization of tetramer-positive cells.
Flow-based intracellular staining methods provide a technically simple and relatively fast method for identifying antigen-responsive cells; however, it is difficult to detect low numbers of antigen-specific cytokine-producing cells at levels significantly above background. The limitation of the assay lies in the fact that low frequency of cytokine producing cells may be indistinguishable above background levels. Addition of immune enhancer reagents, such as antibodies to CD28, are frequently used to augment costimulation in culture conditions but these reagents may bias the results. Success with intracellular staining assays have been most frequently reported in studies evaluating CD4+ T cell responses to viral antigens using PBMC from chronically infected individuals (for example, CMV or HIV). Very limited success has been reported for the detection of ex vivo antigen-specific cells in CD4+ or CD8+ T cells from immunized individuals where the number of circulating antigen-specific cells may be considerably lower than that found in individuals exposed to the infectious agent. Indeed, without using tetramers to select a subpopulation of specific cells for evaluation, it has not been possible to detect low frequencies of antigen-specific cells using flow cytometry for intracellular cytokines. Thus, there is a need in the art for a flow cytometric method of detection low levels of antigen-specific cells with readily available reagents.
In the assays described above, antigen-specific IFN-γ-immune responsiveness is evaluated directly by detecting IFN-γ or IFN-γ producing cells; these assays do not measure the biological response of IFN-γ production from antigen-specific cells as is possible through the amplification effect of MIG. This results in an inability of IFN-γ ELISPOT assays and other detection methods to detect antigen-specific IFN-γ responses at low levels. Accordingly, there is a need in the art for a very sensitive assay for quantitative measure of IFN-γ activity or for detecting antigen-specific IFN-γ-producing cells.
Farber et al., WO 92/10582, published Jun. 25, 1992, teach methods for producing MIG proteins, nucleotide sequences, probes, and antibodies. In a single sentence in the embodiment, the authors implicate that detection of MIG may potentially be used to bioassay for IFN-γ. It is suggested that a sample containing an unknown quantity of IFN-γ is applied to a macrophage or monocytic cell line and the amount of MIG proteins, or MIG messenger RNA which is made in response to the applied IFN-γ may be subsequently quantified. Quantification is taught to be possible by any means known in the art, such as radioimmunoassay, Northern blots, Western blots, enzyme-linked immunoadsorbent assay, etc. The amount of the MIG protein or mRNA produced by the cells in response to the applied IFN-γ would potentially correlate with the amount of IFN-γ in the sample (but no evidence is presented). See WO 92/10582 at pp. 8-9. There are no embodiments in the PCT for using MIG as a bioassay for IFN-γ. More, specifically, there are no embodiments described using the detection of MIG as a marker of antigen-specific immune responsiveness, or antigen-specific IFN-γ production. Moreover, there is no description of how MIG expression could be used as a marker for detecting antigen-specific cells or for detecting antigen-specific IFN-γ producing cells. Thus, there is a need in the art for a more sensitive assay that uses the induction of MIG expression as a marker for antigen specific IFN-γ producing cells or antigen-specific IFN-γ production. While Farber implicates the use MIG as a bioassay for IFN-γ, our application describes induction of MIG expression as a marker for immune responsiveness.
Amichay et al. 1996 (Genes for chemokines HuMig and Crg-2 are induced in protozoan and viral infections in response to IFN-γ with patterns of tissue expression that suggest nonredundant role in vivo. J. Immunol. 157:4511) teach in vivo MIG expression following exposure to a pathogen. In that report, mice were experimentally infected with protozoan or viral pathogens and the level of MIG expression was assessed. Compared to non-exposed controls, induction of MIG expression was noted in various organs and tissues in response to infection, demonstrating that MIG expression is induced following in-vivo infection. Specifically, Amichay demonstrated that infection of mice with different pathogens induced expression of MIG in various organs. Induction of MIG expression following in vivo infection was not observed if they used IFN-γ knockout mice (mice that are genetically incapable of producing IFN-γ). Similarly, injecting mice with IFN-γ also induced MIG expression in various organs as well. These studies did not demonstrate that induction of MIG expression was antigen-specific or genetically-restricted or general to inflammatory stimuli induced by infection, or if it was a consequence of IFN-γ production from antigen-specific cells or if production was specifically mediated by CD8+ T cells, CD4+ T cells, and/or NK cells. Accordingly, there is a need in the art to develop a sensitive and specific assay for IFN-γ activity or other Th-1 cytokine which is based on the detection of MIG or other chemokine as a marker for antigen-specific immune responsiveness or for detecting antigen specific IFN-γ cells.
Recent studies have implicated MIG as an important immune effector molecule in its own right. Like IP-10 and I-TAC, MIG binds to a common receptor, CXCR3, which is known to be expressed on the surface of activated/memory T cells and NK cells (28). These chemokines are induced by a variety of cell types in response to IFN-γ (23, 29, 30). MIG has been shown to enhance NK cell mediated cytotoxicity and to mediate antitumor and antiviral responses in vivo (31, 32). Neutralization of MIG has also been shown to prolong graft survival in vivo (33). Since expression of MIG mRNA can be detected in a variety of different organs following IFN-γ administration, including liver, thymus, lung and spleen, or in the liver and spleen of mice following infection by P. yoelli or T. gondii (7), it is likely that MIG may represent a key mediator of protective immunity.